Excretory-Secretory Products Of Fasciola Gigantica Disrupt The Maturation And Functions Of Buffalo Dendritic Cell Through Regulating Methylation/Hydroxymethylation Progress

Background : Helminthic parasites could possibly disturb the immune microenvironment,reduce or even silence the immune responses in the hosts through releasing excretory-secretory product(ESP),in order to escape from host immune attack during their migration and development.The suppression of host dendritic cell(DC)maturation by helminthic ESP has been proven,while several studies also reported the close connection between DNA methylation/hydroxymethlation and the maturation level of DC.However,whether the Fasciola gigantica is able to disturb DC development in the host through releasing their ESP(FgESP),and if such progress could be modulated by DNA methylation/hydroxymethylation,are still unclear.This thesis mainly focuses such question: if the impacts of FgESP on the development and functions of DC in the natural host,is based on the alteration of DNA methylation/hydrxymethylation.And the potential mechanisms of immune regulation are also discussed.Methods: The in vitro induction and culture methods of buffalo peripheral blood derived DC were established and optimized.These buffalo DCs were co-cultured with FgESP in vitro,the changes of MHC Ⅱ,CD83,IL-6 and IL-12 mRNA in DCs were determined by quantitative real-time polymerase chain reaction(q RTPCR).Histone methylation detection kits were applied to detect the changes of H3K4,H3K9 and H3K27 histones.Genomic DNA methylation/hydroxymethylation level in buffalo DCs was measured and analyzed through the(h)MeDIP-Seq high-throughput system and bioinformatics analyses.The mRNA levels of selected genes of our research interests were quantitated by qRT-PCR.Co-immunoprecipitation(Co-IP)assay was employed to detect any components of FgESP directly binding with DNMT1 or TET1 protein in buffalo DC.The subcellular localization of DNMT1 or TET1 protein was predicted by bioinformatics softwares.Co-localization was assayed by cellular immunofluresence in the aim to discuss the possibility of direct interaction between FgESP and DNMT1/TET1 protein in buffalo DC.To further investigate the possibility of indirect interaction between FgESP and DNMT1/TET1 protein,DNMT1 and TET1 genes were knock-down by usingRNAi technology,then the changes of CD1 a,CD40,CD80,CD83,CD86 and MHC Ⅱ mRNA in DCs were determined by qRT-PCR,whereas the cytokine levels of IL-6,IL-10,IL-12 and TNF-α were measured by enzyme-linked immuno sorbent assay(ELISA).Results: The morphological feature and molecular expression pattern of buffalo peripheral blood derived DCs conform to basic characteristics of immature DC(im DC).200 μg/ mL of FgESP was shown to induce upregulated expression of MHC Ⅱ,CD 83 and IL-12 mRNA but downregulated expression of IL-6 mRNA,suggesting the suppression of DC maturation.The methylation level of H3K4 was found decreased while that of H3K9 was increased in DCs after FgESP treatment,hinting the possible presence of DNA methylation.The analysis of(h)MeDIP-Seq has identified 5432 of genes with significantly changed 5-methylcytosine(5-m C)level and 360 of genes with 5-significantly changed 5-hydroxymethylcytosine(5-hm C)level,while 111 of genes with significance of both 5-m C and 5-hm C alteration.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses indicated that these differentiate expressed genes were highly enriched in pathways related with immune responses,and partially enriched in cancer-related pathways.The result of qRT-PCR validated that highly methylated genes TLR2,TLR4 and IL-12 B also possessed reduced mRNA levels or downtrend,while highly hydroxymethylated genes TNF-αshowed significant upregulation of mRNA level,suggesting a direct connection between DNA methylation/hydroxymethylation and suppression of DC maturation mediated by FgESP.No specific band of bonding between FgESP component and DNMT1 or TET1 protein from buffalo DC was detected by Co-IP test.Similarly,the Cy3 fluorescent dye-prelabelled FgESP was found to spread on the membrane of in the cellular plasma,but not on the nuclei.This seems very unlikely that FgESP would directly react with either buffalo DNMT1 or TET1 protein which was predicted to localize in the nuclei.FgESP was also found to reduce the ability of buffalo DC in producing IL-6 and IL-12 cytokines afterRNAi of DNMT1 or TET1,as an indirect evidence to confirm the link between FgESP and buffalo DC development and functions.Conclusions: According to the analysis of experimental results,it is preliminarily believed that FgESP affects the development,cellular immune responses,and histone methylation level of buffalo DC.And,we,for the first time,have also reported the whole genomic DNA methylation/hydoxymethylation profile for the interaction between FgESP and buffalo DC using a high-throughput technology.The involvement of DNMT1 and TET1(key methylation/hydoxymethylationrelated molecules)in the interaction between FgESP and host DC,was preliminarily investigated and discussed.Our data has showed that FgESP could suppress the development and functions of buffalo DC to some extent,possibly rely on TLR signaling and the relevant gene expression.DNMT1-and TET1-related pathways might also take part in the regulation of IL-6 and IL-12 production,respectively.These research results will provide important evidences as the reference for further studies investigating and explaining how FgESP modulates DC development and functions thereby helps the parasite to escape from the immune attacks.